1. Field of the Invention
The present invention relates to water-soluble synthetic glycosyl orthoesters of vitamin D, and their use in the regulation of calcium metabolism.
2. Description of the Background Art
Vitamin D.sub.3 deficiency, or disturbances in the metabolism of vitamin D.sub.3 cause such diseases as rickets, renal osteodystrophy and related bone diseases, as well as, generally, hypo- and hyper-calcemic states. Vitamin D.sub.3 and its metabolites are therefore crucial in maintaining normal development of bone structure by regulating blood calcium levels.
Vitamin D.sub.3 is rapidly converted to 25-OH-D.sub.3 in the liver. In response to hypocalcemia, 25-OH-D.sub.3, the major circulating metabolite of the vitamin, undergoes further metabolism in the kidney to 1,25-(OH).sub.2 D.sub.3. 1,25-(OH).sub.2 D.sub.3 acts more rapidly than either D.sub.3, or 25-OH-D.sub.3. Additionally, the dihydroxy form of the vitamin is 5-10 times more potent than D.sub.3, and about 2-5 times more potent than the monohydroxy form of the vitamin, in vivo, provided it is dosed parenterally and daily (Napoli, J. L. and Deluca, H. F., "Blood Calcium Regulators" and references cited therein in: Burger's Medicinal Chemistry, 4th Ed., part II, edited by Manfred Wolf, Wiley-Interscience, 1979, pp. 725-739).
Vitamin D.sub.2, vitamin D.sub.3 or their metabolites which are hydroxylated at positions 1; 1,25; 1,24,25; 24,25; 25,26; or 1,25,26 are water-insoluble compounds. When a drug is relatively insoluble in an aqueous environment or in the gastrointestinal lumen, post-administration dissolution may become the rate-limiting step in drug absorption. On the other hand, with water-soluble drugs, dissolution occurs rapidly and thus facilitates transport through blood and to the site of activity. It would therefore be desirable to provide a form of vitamin D (D.sub.3 or D.sub.2) which is hydrophilic and/or water-soluble, yet preserves the normal biological properties of the water-insoluble drug.
The extracts from the leaves of a South American plant, Solanum malacoxylon (hereinafter "S.m."), have been demonstrated to contain a water-soluble principle which is different than 1,25(OH).sub.2 D.sub.3 and which, upon treatment with glycosidase enzymes yields 1,25(OH).sub.2 D.sub.3, plus a water-soluble unidentified fragment. (See, for example, Haussler, M. R., et al., Life Sciences, Volume 18: 1049-1056 (1976); Wasserman, R. H., et al., Science 194: 853-855 (1976); Napoli, J. L., et al., The Journal of Biological Chemistry, 252: 2580-2583 (1977)).
A very similar water-soluble principle, which upon treatment with glycosidases also yields 1,25-dihydroxy vitamin D.sub.3, is found in the plant Cestrum diurnum (hereinafter "C.d."); Hughes, M. R., et al., Nature, 268: 347-349 (1977)). The water soluble extracts for S.m. or C.d. have biological activity which is similar to that of 1,25-dihydroxy vitamin D.sub.3.
The only evidence concerning the structure of the water-soluble fragment released during glycosidase treatment of the water-soluble principles from these plants is indefinite. The authors of the aforementioned publications have concluded that the structure is probably a glycoside, on the basis of enzymatic evidence, the water-solubility, and the use of chemical detection reagents (Peterlik, N. and Wasserman, R. H., FEBS Lett. 56: 16-19 (1973)). Humphreys (Nature (London) New Biology 246: 155 (1973)), however, has cast some doubt on this conclusion since he demonstrated that the Molisch carbohydrate test was negative for the principle.
Since it is known that the molecular weight of the water-soluble vitamin D.sub.3 -containing principle, prior to enzymatic release, is considerably greater than 1000 (Humphreys, D. J., Nature (London) New Biology 246: 155 (1973)), the molecular weight of the water-soluble conjugated fragment released by enzymatic hydrolysis can be calculated to be considerably greater than 584, the molecular weight of dihydroxy vitamin D.sub.3 being 416. Thus, if the water-soluble fragment released by enzymatic hydrolysis were in fact a glycoside, it would contain more than 3 glycosidic (glycopyranosyl or glycofuranosyl) units.
Moreover, the results of enzymatic release are fully consistent with a wide variety of structures. For example, Haussler, M. R., et al., Life Sciences 18: 1049-1056 (1976) disclose the use of mixed glycosidases derived from Charonia lampus to hydrolyze the water-soluble principle. This enzyme is really a mixture of enzymes, as follows (Miles Laboratories, 1977 catalog): .beta.-glucosidase (11 units), .alpha.-mannosidase (33 units), .beta.-mannosidase (5.2 units), .alpha.-glucosidase (4.8 units), .beta.-galactosidase (44 units), .alpha.-galactosidase (26 units), .alpha.-fucosidase (24 units), .beta.-xylosidase (8.2 units), .beta.-N-acetylglucosaminidase (210 units), .alpha.-N-acetylgalactosaminidase (41 units), and .beta.-N-acetyl-galactosaminidase (25 units). Peterlik, M., et al. (Biochemical and Biophysical Research Communications, 70: 797-804 (1976)) in their study of the S.m. extract with .beta.-glucosidase (almond) from Sigma Chemical Company, utilized an enzyme that also contained .beta.-D-galactosidase, and .alpha.-D-mannosidase activities (Sigma Chemical Company, February 1981 Catalog; see also, Schwartz, J., et al., Archives of Biochemistry and Biophysics, 137: 122-127 (1970)).
In sum, the results observed by these authors are consistent with a wide range of structures, none of which have been well characterized but which, even if proven to be glycosides, contain at least more than 3 glycosidic units per vitamin D unit.
Holick, et al., U.S. Pat. No. 4,410,515 describe water-soluble glycoside derivatives of Vitamin D which are biologically active. Furst, et al., Helv. Chim. Acta, 66: 2093 (1983) have also synthesized Vitamin D glycopyranosyl derivatives.
A need, however, continues to exist for other well-defined, well-characterized water-soluble forms of vitamin D, which will be hypocalcemically active and maintain calcium and phosphorus homeostasis.